Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. Frequently, key intracellular signaling proteins move from one subcellular location to another (e.g., from cytoplasm to plasma membranes or from cytoplasm to nucleus) in response to extracellular stimuli. Incorrectly localized proteins can prevent completion of a particular signaling pathway or can cause unregulated signaling, such as uncontrolled cell growth. Understanding how cellular proteins come together at specific times and subcellular sites will lead to insight into ways to block inappropriate signaling in disease states. The heterotrimeric (alphabetagamma) G proteins act as molecular switches to relay information from activated cell-surface receptors to appropriate intracellular effectors. One family of G protein a subunits, consisting of alpha12 and alpha13, can activate the Rho family of small GTPases. Rho, in turn, activates signaling pathways that stimulate cell growth and morphological changes. A large family of Rho guanine nucleotide exchange factors (GEF) activates Rho, and a sub-family, termed RGS-RhoGEFs, and consisting of p115-RhoGEF, PDZ-RhoGEF, and LARG, function as direct links between alpha12/13 and Rho. However, the mechanisms responsible for activation of the RGS-RhoGEFs by alpha12 and alpha13 are poorly understood. The main objectives of this application are to elucidate the mechanisms that control subcellular localizations of the RGS-RhoGEFs, determine how differential and regulated subcellular localization of the RGS-RhoGEFs contribute to signaling function, and define the interactions between alpha12/13 and the RGS-RhoGEFs. These objectives will be addressed through the following specific aims: (1) Define subcellular localization of LARG, and determine changes in localization in response to activated Ga and GPCRs; (2) Define the functional significance of the actin-binding domain of PDZ-RhoGEF; (3) Define the involvement of Rho in alpha13- dependent PM recruitment of p115-RhoGEF; (4) Define interactions between RGS-RhoGEFs and alpha13-alpha12/13 or alphaq, and determine signaling functions of RhoGEF binding-defective mutants of alpha12/13. This research will utilize cultured mammalian cells as the model systems and will employ cell biology and biochemical techniques to examine the central hypothesis that subcellular localization plays a critical role in understanding RGS-RhoGEF function and how alpha12/13 activate Rho signaling pathways through p115-RhoGEF, PDZ-RhoGEF and LARG.
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